Synthesis of these molecules on a solid support, in particular the synthesis of peptides, is well known from the work of MERRIFIELD in 1963 and is widely used. It essentially comprises sequentially assembling amino acids to form a peptide, by fixing one end of the chain to an insoluble support such as a polymer. Once the desired sequence of amino acids has been sequentially assembled, this chain is cleaved to separate the peptide from the support and release it in solution.
The reactions brought into play in this synthesis are essentially reactions of coupling of amino acids, performed alternately with reactions of stripping desired functions of the amino acids, and washing and rinsing operations between the end of one reaction and the beginning of the next reaction.
It is essential in this synthesis that each coupling or stripping reaction be performed to 100%, or almost. The only means that currently makes it possible to ensure this is to interrupt the synthesis to carry out a test or an assay. This considerably prolongs the length of a synthesis and prevents it from being automated. Moreover, the tests used do not always permit verification of whether a reaction has taken place completely, so that in the event of uncertainty the reaction must be restarted for increased certainty.
Finally, in these classic synthesis processes it is generally necessary to use excess ingredients and coupling reagents, for many reasons (inaccessibility of some of the reaction sites of the polymer, weak attachment of the ingredients on the polymer or on an ingredient already fixed to the polymer, and so forth).
All these disadvantages mean that classic peptide synthesis processes have a low yield, of the order of 20 to 30%, as a function of the length of the peptide chain, and that the cost of peptides is extremely high (sometimes more then 1000 FF/milligram of peptide).
In an attempt to reduce these disadvantages, the proposal has already been made to monitor the coupling reactions with ultraviolet spectroscopy, which is believed to detect a peak signal corresponding to the end of the coupling reaction. However, the results have shown that this method was not very reliable and could lead to errors or major difficulties in interpretation.
It has also been proposed that the electrical conductance of the reaction medium be measured. However, this medium is particularly heterogeneous, and in this process considerable background noise in which the useful signal is embedded is detected, which makes the process difficult to exploit.
The object of the invention is essentially to provide a simple, effective and particularly reliable solution to the problem of monitoring coupling and stripping reactions performed in peptide, polynucleotide or oligosaccharide syntheses.
It is based on the fact that each coupling or stripping reaction causes a variation in temperature of the reaction medium, and that under certain conditions it may be assumed that the reaction is completed once this variation in temperature becomes substantially zero.
However, it is virtually impossible directly to measure this variation in temperature, because it is much slighter (by one or more orders of magnitude) than the variations in temperature due to parasitic reactions in the reaction medium, notably reactions between solvents.
One of the essential merits of the present invention is accordingly that of successfully detecting the very slight variation in temperature of the reaction medium that accompanies a coupling or stripping reaction, and of monitoring the development of this variation in temperature very precisely in order to detect the end of the coupling or stripping reaction surely and reliably.